This invention relates to N-acylated glucosamine derivatives; methods of treating arthritis, particularly osteoarthritis and inflammatory arthritis, enhancing cartilage formulation, enhancing chondrocyte cell proliferation and glycosaminoglycan production in a mammal with said derivatives; and pharmaceutical compositions comprising said N-acylated glucosamine derivatives.
It is known that glycoconjugates play an important role in many biological processes. The carbohydrate groups confer important physical properties such as conformational stability, protease resistance, charge and water-binding capacity; and biological recognition, where sequence diversity provides signals for protein targeting and cellxe2x80x94cell interactions (Paulson 1989). The glycoconjugates of connective tissue matrices consist of hexosamines that are N-acetylated. However, the function of the N-acetyl moiety is not known.
The two major forms of arthritis in mammals are inflammatory arthritis, such as rheumatoid arthritis (RA), and osteoarthritis (OA), a progressive, degenerative loss of cartilage often secondary to mechanical stress, aging, dysplastic conditions and/or injury. Pain in OA is usually treated with NSAIDS (non-steroidal anti-inflammatory drugs). Inflammation and pain in RA is treated with NSAIDS, with new COX-2 inhibitors (also NSAIDS) and also with anti-metabolites such as methotrexate. Other immunomodulators in clinical use or trials include interleukins and TNF receptor antagonists. Glucosamine is a popular non-prescription, neutraceutical treatment for pain in OA. Since RA and OA have different pathologies, it is not obvious that a treatment for one should result in a treatment for the other. A recent review, J. Rheumatol. (1999) 26:11-Anastassiades T., notes that many reports of glucosamine/OA clinical trials indicate positive findings but the mechanism of action is unknown.
When glucosamine is given even in very large doses to humans it is quickly cleared from circulation to the point that serum levels cannot be detected after oral or IV administration.
Glucosamine derivatives have been examined as potential therapeutic agents. When compared to glucosamine, N-acetylglucosamine (GluNac) has been shown to have a longer half-life when administered to humans Clin. Ther. (1996) 18:1184 in polyvalent or monvalent form, but no efficacy data were recorded. This reference proposes GluNac as a potential therapeutic for OA but it did not propose any rationale for therapy apart from serum levels.
A number of patents for example, U.S. Pat. No. 4,314,999, U.S. Pat. No. 5,696,098 and European Patent 356275 discuss chemical modifications of amino sugars that are structural components of oligosaccharides or polysaccharides i.e. covalently bound, but are not compounds of the present invention which are chemical modifications of a monosaccharide i.e. a single sugar molecule such as glucosamine.
Despite theories of chondroprotective actions, when given in vitro to bovine chondrocytes, glucosamine does not support growth or even survival of chondrocytes. In the presence of glucosamine, bovine chondrocytes grow much more slowly than in the absence of glucosamine, in culture which suggests it is not acting as a chondroprotective agent. Biochem. Pharmacol. (1973) 22:3018-Anastassiades T. discloses that a propionyl derivative of glucosamine actually inhibits the incorporation of labeled glucosamine into mucopolysaccharides, the older term for glycosaminoglycans and, accordingly, this reference teaches that N-propionyl glucosamine should inhibit cartilage formation.
Proteoglycan (PG) consists of a non-collagenous protein core to which long-chain polysaccharides (glycosaminogylcans, GAGs) are linked. PG is a key component of cartilage which accounts for its biomechanical properties. Type II collagen is the other principle component of cartilage. These two components are thus often used, alone or in combination, as in vitro surrogate markers for cartilage synthesis and degradation. Beekman R. (1998) Articular chondrocytes: synthesis and MMP-mediated degradation of extracellular matrix. Thesis from the Gaubius Laboratory of TNO Prevention and Health, Leiden, The Netherlands (ISN 90-9011354-1).
It is an object of the present invention to provide a method of treating arthritis, particularly, osteoarthritis, inflammatory arthritis, traumatic arthritis, degenerative arthritis and dysplastic arthritis.
It is a further object to provide a method of alleviating the unwanted symptoms of arthritis of joint stiffness and restricted mobility.
It is a further object to provide a method of enhancing cartilage formation.
It is a further object to provide a method of enhancing mammalian chondrocyte cell growth.
It is a further object to provide a method of enhancing the production of glycosaminoglycan in a mammal.
It is a further object to provide the use of N-acylated gluosamine derivatives for optimization of a bovine cartilage (BAC) growth assay for the stimulatory effects of these compounds. Accordingly, in one aspect the invention provides a method of treatment of arthritis, particularly, of osteoarthritis and inflammatory arthritis in a mammal comprising administering to said mammal an effective amount of a N-acylated-2-glucosamine derivative of the general formula (I): 
wherein R is an alkyl radical of the general formula CnH2n+1 wherein n is selected from 2-12, and pharmaceutically acceptable salts, esters and glucosides thereof.
Preferably, n is selected from 2-5 and more preferably 3.
In a further aspect the invention provides a method of enhancing chondrocyte cell proliferation comprising treating a population of chondrocyte cells with an effective amount of a N-acylated-2-glucosamine derivative as defined hereinabove.
In this specification, all references to glucosamine and its N-acylated derivatives means 2-amino-2-deoxy-D-glucose and its N-acylated derivatives (DGlcNs).
In this specification the term xe2x80x9cmammalsxe2x80x9d includes, but is not limited to, human beings, particularly, dogs.
Specifically, the preferred compounds are:
N-Butyryl-D-glucosamine (2-n-Butanamido-2-deoxy-D-glucopyranose) (GlcNbu);
N-Valeryl-D-glucosamine (2-n-Pentanamido-2-deoxy-D-glucopyranose) (GlcNva);
N-Capryl-D-glucosamine (2-n-Hexanamido-2-deoxy-D-glucopyranose) (GlcNca) and branched alkyl isomers, e.g. secondary and tertiary analogues thereof.
The most preferred compound is N-butyryl-D-glucosamine, of the formula II: 
In a further aspect, the invention further provides a method for enhancing the production of glycosaminoglycan by the treatment of chondrocytes with an effective amount of a N-acylated-2-glucosamine as hereinabove defined.
In still a further aspect, the invention provides a method of enhancing cartilage growth and formation in a mammal by administering to said mammal an effective amount of a N-acylated glucosamine as hereinabove defined.
In yet a further aspect, the invention provides a diagnostic test involving the use of labeled N-derivitized glucosamine monomers to monitor growth of cartilage in subjects receiving treatment since the compounds of use in the present invention are incorporated into growing cartilage.
The N-acylated derivatives may be administered to a mammal by, for example, one of the following methods, namely, orally, intravenously, inter-arterially, dermally or subcutaneously.
The derivative may be typically administered in a suitable vehicle, in which the active ingredient is either dissolved or suspended in a liquid and which permits the N-acylated glucosamine to be delivered to the arthritic site from the bloodstream or transdermally. Solution compositions would be, typically, alcohol solutions, dimethyl sulfoxide solutions, or aqueous solutions containing, for example, polyethylene glycol. Such vehicles are well-known in the art, and useful for the purpose of delivering active ingredients to the site of action. To work, the active ingredient must be administered in a solvent that would prevent them from precipitating in the otherwise aqueous environment of the bloodstream. The solvent dimethylsulfoxide is one such solvent.
It will be understood by the person skilled in the art that the active N-acylated glucosamines as hereinbefore defined should be present in respective, effective amounts to (a) alleviate the symptoms of arthritis, (b) enhance cartilage formation in a mammal, (c) enhance chondrocyte cell proliferation, (d) enhance production of glycosaminoglycan; and (e) alleviate the symptoms associated with arthritis of joint stiffness and restricted mobility.